Department of Pharmaceutical Sciences, College of Pharmacy, A323A ASTeCC Bldg., University of Kentucky, Lexington, Kentucky 40506

Transcription

1 Liposome Transport of Hydrophobic Drugs: Gel Phase Lipid Bilayer Permeability and Partitioning of the Lactone Form of a Hydrophobic Camptothecin, DB-67 VIJAY JOGUPARTHI, TIAN-XIANG XIANG, BRADLEY D. ANDERSON Department of Pharmaceutical Sciences, College of Pharmacy, A323A ASTeCC Bldg., University of Kentucky, Lexington, Kentucky Received 15 January 2007; revised 3 June 2007; accepted 6 June 2007 Published online in Wiley InterScience ( DOI /jps ABSTRACT: The design of liposomal delivery systems for hydrophobic drug molecules having improved encapsulation efficiency and enhanced drug retention would be highly desirable. Unfortunately, the poor aqueous solubility and high membrane binding affinity of hydrophobic drugs necessitates extensive validation of experimental methods to determine both liposome loading and permeability and thus the development of a quantitative understanding of the factors governing the encapsulation and retention/ release of such compounds has been slow. This report describes an efflux transport method using dynamic dialysis to study the liposomal membrane permeability of hydrophobic compounds. A mathematical model has been developed to calculate liposomal membrane permeability coefficients of hydrophobic compounds from dynamic dialysis experiments and partitioning experiments using equilibrium dialysis. Also reported is a simple method to study the release kinetics of liposome encapsulated camptothecin lactone in plasma by comparing the hydrolysis kinetics of liposome entrapped versus free drug. DB-67, a novel hydrophobic camptothecin analogue has been used as a model permeant to validate these methods. Theoretical estimates of DB- 67 permeability obtained from the bulk solubility diffusion model and the barrierdomain solubility diffusion model are compared to the experimentally observed value. The use of dynamic dialysis in drug release studies of liposome and other nanoparticle formulations is further discussed and experimental artifacts that can arise without adequate validation are illustrated through simulations. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97: , 2008 Keywords: bilayer; kinetics; cancer chemotherapy; permeability; encapsulation; liposomes; mathematical model; membrane transport; passive diffusion/transport INTRODUCTION Correspondence to: Bradley D. Anderson (Telephone: , Ext 235; Fax: ; bande2@ .uky.edu) Journal of Pharmaceutical Sciences, Vol. 97, (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association Silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin (DB-67, Scheme 1) is a novel anti-cancer agent with superior blood stability and potent anti-cancer activity compared to other camptothecin analogues. 1,2 It has been recently approved by the FDA for phase I clinical studies. 3 The biologically active lactone forms of camptothecins undergo ph dependent hydrolysis in solution to the inactive ring opened carboxylate forms. 4 6 The carboxylate form is the favored species at equilibrium at physiological ph. 6 In human blood this equilibrium may be further shifted towards the carboxylate due to its preferential binding to serum albumin Efforts to synthesize camptothecins that remain in their active lactone form in blood resulted in the development of potent, 400 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008

2 LIPOSOME TRANSPORT OF HYDROPHOBIC DRUGS 401 Scheme 1. Equilibrium between DB-67 lactone (left) and DB-67 carboxylate (right). Rate constants k o and k c are the lactone ring opening and closing rate constants. highly lipophilic analogues with improved blood stability but poor water solubility. 12 DB-67, along with other camptothecin analogues such as karenitecin and gimatecan, represent this new generation of blood stable but water insoluble camptothecins. 13,14 Novel formulation strategies are required to enable the delivery of these highly potent anti-cancer agents. Carriers that improve delivery of these agents to tumor tissue are also needed to diminish their toxic side effects. Liposome technology has significant potential to improve formulation (e.g., solubility and stability) and tumor delivery-related issues that hinder the clinical advancement of these novel camptothecins. Lipid bilayer association has been previously found to improve solubility and stability of camptothecins Liposomes are known to preferentially accumulate in tumor tissue after an i.v. injection and thus liposomal encapsulation offers the potential for improved antitumor specificity. 18 Therefore, liposomal formulations are currently being investigated for various camptothecin analogues, several of which are in preclinical or clinical trials One current strategy for liposomal encapsulation of camptothecins exploits the ph dependent lipid bilayer association of those compounds having an ionizable amine on the A or B- ring. 21,23 25 The ring-closed lactone has a greater membrane binding constant than the ring-opened carboxylate and therefore a low intraliposomal ph stabilizes the biologically active lactone form in lipid bilayers. 15,21,23 25 A variety of loading techniques (e.g., ph, ammonium sulfate or ion gradient loading, metal ion complexation, etc.) developed to improve encapsulation efficiency of amphiphilic amines are applicable to aminecontaining analogs or prodrugs of camptothecins. 21,23,26 29 However, these techniques do not improve the loading efficiency of the lactone forms that lack an ionizable amine group such as DB-67 and camptothecin itself. Another important consequence of the lack of an ionizable cationic functional group is the poor liposomal retention of the neutral analogs. For example, 28% of the amine-containing camptothecin, lurtotecan, was retained in liposomes 4 h after an i.v. injection 20 while only 1% of the neutral camptothecin, SN-38, remained in the circulation 4 h after administration of a liposomal formulation. 22 Premature leakage of the encapsulated drug fails to take advantage of the passive tumor targeting of liposomes and increases the potential for side-effects to healthy tissue. To overcome such delivery-related issues with DB-67, efforts are underway in our laboratories to develop novel cationic and anionic prodrug strategies to improve liposomal encapsulation efficiency during formulation and drug retention in liposomes in vivo. A prerequisite to understanding the factors governing liposomal encapsulation, retention and release in vitro and in vivo is the knowledge of bilayer permeability of the biologically active and presumably bilayer permeating camptothecin lactone. To our knowledge, systematic studies of the lipid bilayer permeability of camptothecins including DB-67 have not been previously reported. The objective of the current work was to understand the gel phase lipid bilayer permeability of the lactone form of DB-67 in vitro both in aqueous buffer and plasma since pegylated liposomes with a rigid gel phase bilayer have better retention of encapsulated drug in vivo. 18 DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008

3 402 JOGUPARTHI, XIANG AND ANDERSON Previously, gel filtration and ultrafiltration methods have been used in these laboratories to study the bilayer permeability of various compounds However the high lipophilicity (clog P ¼ ) (calculated from Advanced Chemistry Development (ACD) Labs software) of DB-67 precluded the use of these methods due to ultrafilter membrane binding and the difficulty of maintaining good sink conditions in transport experiments. Therefore, a dynamic dialysis method to study membrane permeability of hydrophobic solutes has been developed and validated. A mathematical model has also been developed to calculate the bilayer permeability coefficients for hydrophobic solutes using dynamic dialysis and simulations were performed to identify situations where the method is suitable for quantifying liposome release kinetics of hydrophobic compounds. A theoretical estimate of the permeability coefficient for gel phase bilayer permeation of DB-67 was obtained using the bulk solubility-diffusion model and the recently developed barrier-domain solubility-diffusion model 33 and compared to the experimental value. The kinetics of drug release from liposomes in vivo may be influenced by alteration of the bilayer barrier properties by serum proteins that adsorb to membranes 34 and by the presence of transbilayer ph gradients. The hydrolysis kinetics of liposome entrapped versus free DB-67 lactone in rat plasma were monitored in the studies described herein to estimate the rate constant for drug release from liposomes in the presence of plasma proteins. MATERIALS AND METHODS Materials 1,2-Distearoyl-sn-glycero-3-phosphatidylcholine (DSPC, >99% purity) and 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (m-peg DSPE, MW ¼ 2806, >99% purity) were purchased as powders from Avanti Polar Lipids (Alabaster, AL) while DB-67 was obtained from Novartis Pharmaceuticals Corporation (East Hanover, NJ). Dialysis tubes (Float-A-Lyzer 1, MWCO: ) and precut dialysis membrane discs (MWCO: ) were purchased from Spectrum Laboratories (Rancho Dominguez, CA). Sephadex TM G-25 M pre-packed size exclusion columns were obtained from GE Healthcare Bio-sciences JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008 Corporation (Piscataway, NJ). 1, 9 decadiene was obtained from Sigma Aldrich Fine Chemicals (St. Louis, MO). All other reagents were purchased from Fisher Scientific (Florence, KY). Preparation of Liposomes Pre-weighed lipids, DSPC and m-peg DSPE (95:5 mol%), were dissolved in chloroform and distributed into test tubes. Chloroform was subsequently evaporated under a stream of nitrogen gas and the residue was vacuum-dried (****408C, 6 h). The dried lipid film was hydrated with acetate (85 mm, ph ¼ 4 4.2) or citrate (100 mm, ph ¼ 4.5) buffer in a608c water bath with vigorous shaking to obtain a 16 mg/ml (w/v) lipid suspension. The lipid suspension was extruded (10, 608C) through two stacked 200 nm polycarbonate membranes (Nuclepore, Pleasanton, CA) using an extrusion device (Liposofast 1, Avestin, Canada) to obtain unilamellar vesicles. Liposomes prepared with acetate buffer were used in partitioning and release experiments in aqueous buffers and liposomes prepared with citrate buffer were used for release kinetics in rat plasma. DB-67 was loaded into liposomes by adding 2 5 ml of a stock solution of DB-67 (1 100 mm) in DMSO to ml of blank liposome suspension and incubating at 608C for 2 h. Following their preparation, both blank and drug-loaded liposomes were allowed to cool to room temperature for 3 h and stored at 58C until the beginning of transport experiments. All vesicle preparations were used within a week. Particle size by dynamic light scattering (DLS, Malvern Zetasizer-3000, Malvern Instruments Ltd, Malvern, UK) and ph were monitored both after preparation and prior to use. Sampling Procedures Previous studies in these laboratories demonstrated that DB-67 adsorbs to various surfaces including glassware, pipette tips, ph electrodes, etc. 35 A single methanol wash was found to recover most of the adsorbed drug. 35 Therefore, all pipette tips used for sampling in partitioning and permeability studies were subjected to a methanol wash and the washings were transferred to the same vial as the sample. DOI /jps

4 LIPOSOME TRANSPORT OF HYDROPHOBIC DRUGS 403 Determination of 1, 9-Decadiene and Lipid Bilayer/Water Partition Coefficients The 1, 9-decadiene/water partition coefficient for DB-67 was determined by adding a 1 ml stock solution of DB-67 in DMSO to ml of 1,9- decadiene pre-washed with 0.04 N HCl to obtain a nm concentration. Acetate buffer (1 ml, ph ¼ 4) was mixed with 1 ml of the decadiene containing DB-67 and vigorously vortexed (2 h at 378C. The mixture was allowed to phase separate overnight at 378C and the aqueous phase was analyzed for DB-67 concentration. A sample was also collected from the initial decadiene phase prior to addition of the acetate buffer. Samples were diluted in methanol before HPLC analysis. The liposome membrane/water partition coefficient of DB-67 lactone was determined by equilibrium dialysis (1 ml Teflon 1 cells, Equilibrium Dialyzer, Spectrum Laboratories). Blank liposome suspension (1 ml, mg lipid/ ml) was loaded into the donor compartment and dialyzed at 378C against 1 ml of DB-67 (5 20 nm) in acetate buffer in the receiver compartment. At equilibrium (24 48 h), 100 ml of liposome suspension and drug solution from the donor and receiver compartments, respectively, were withdrawn and transferred to an HPLC vial followed by a syringe wash (200 ml of acidified methanol) to recover adsorbed drug. Samples were stored at 258C until HPLC analysis for DB-67. At equilibrium, the total concentration in the donor compartment containing liposomes can be related to concentration in the bilayer membrane and aqueous phase as follows: C d V d ¼ C w V w þ C m V m (1a) where C d is the total drug concentration in the donor compartment, C w and C m are the molar solute concentrations in the aqueous phase and membrane, respectively, and V d, V w and V m are the respective volumes. The volume partition coefficient, K p can be obtained from dialysis measurements by combining Eq. (1a) with the definition of K p : K p ¼ C m ¼ C dv d C w V w ffi C dv d C r V w (1b) C w C w V m C r V m where C r is the drug concentration in the receiver compartment. The volume of the membrane phase was estimated from the lipid concentration and volume occupied per lipid molecule (estimated from bilayer thickness and area per head group). 36,37 The aqueous volume entrapped in each liposome was estimated from the particle size and bilayer thickness. Determination of Liposome Release Kinetics in Buffers Liposome entrapped drug was separated from free drug by passing through a Sephadex 1 G-25 column pre-equilibrated to a required ph (4 4.2) by washing with 50 ml of the acetate buffer. To validate the separation of liposome entrapped from free DB-67, 0.5 ml of drug loaded liposomes were loaded onto a Sephadex 1 column followed by 35 ml buffer. Eluent fractions (0.2 or 0.5 ml) were analyzed for DB-67 concentration by HPLC and relative lipid concentration by DLS (light scattering intensity of unilamellar vesicles was found to be linearly proportional to lipid concentration over a range of mm). DB-67 release kinetics experiments were initiated by passing 75 ml of drug loaded liposome suspension through a Sephadex 1 column followed by 5 ml of acetate buffer loaded in 1 ml increments. A 5 ml portion of eluent was collected (containing liposome suspension), further diluted to 7.5 ml, and immediately transferred to a dialysis tube preconditioned in deionized water for 15 min and in acetate buffer for 30 min and dialyzed against 1 L of the same buffer at 378C. At various time points 100 ml of liposome suspension was withdrawn from inside the dialysis tube using a precision pipettor and transferred to a polypropylene vial. The pipette tip was washed with 100 ml acidified methanol and the wash solution was transferred to the same vial. Samples were stored at 258C until HPLC analysis for DB-67 concentration. Blank liposome suspensions at a lipid concentration similar to that in transport experiments were spiked with DB-67, immediately placed in a dialysis tube, and dialyzed against 1 L acetate buffer in order to determine the influence of the dialysis membrane transport step on the apparent rate of DB-67 efflux from liposomes. Samples were collected at various time points and processed similarly to DB-67 loaded liposome samples. Determination of Kinetics of DB-67 Release from Liposomes and Rat Plasma Hydrolysis DB-67 efflux from liposomes was also investigated in rat plasma using an indirect method in which the hydrolysis kinetics of DB-67 were monitored DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008

5 404 JOGUPARTHI, XIANG AND ANDERSON in the presence or absence of liposome encapsulation. Kinetic studies of the hydrolysis of the lactone in plasma were initiated by adding a 1 2 ml stock solution of DB-67 in DMSO to 4 ml of rat plasma in ph 7.4 phosphate buffered saline (PBS) to obtain a concentration of nm. Samples were incubated at 378C and 50 ml of plasma was withdrawn at various times and added to 150 ml of cold ( 258C) methanol:acetonitrile (2:1; (v/v)) in a microcentrifuge tube. The mixture was immediately centrifuged at ( 98C, rpm, 3 min) and the supernatant was stored at 258C until HPLC analysis. All samples were kept on dry ice during sample dilutions and transfers from freezer to HPLC sample chamber. Validations of the DB-67 extraction efficiency and quenching of the hydrolysis reaction were performed by spiking blank rat plasma with either DB-67 lactone or carboxylate extractant solution (in cold methanol acetonitrile) and processing spiked plasma similar to that of the reaction samples. Studies of the hydrolysis kinetics of DB-67 in plasma when added in liposomally entrapped suspensions were initiated after separating liposome entrapped from free drug by sizeexclusion chromatography (Sephadex 1 columns equilibrated with ph 7.4 PBS). A ml aliquot of the eluent containing drug-loaded liposomes was added to 4 ml of rat plasma and incubated at 378C. Samples were collected at various times and processed similarly to those generated in studies of free DB-67 hydrolysis (vide supra). HPLC Analyses Samples from liposome partitioning and release experiments were analyzed for DB-67 concentration by HPLC. 16 A Waters Alliance 2690 separation system coupled to a Waters fluorescence detector (M474) was employed with excitation and emission wavelengths at 380 and 560 nm, respectively. A Waters Symmetry 1 C 18 (5 mm) column ( mm) and guard column ( mm) were used with a mobile phase (48% acetonitrile:52% (v/v) of 2% (ph ¼ 5.5) triethylamine acetate buffer) flow rate of 1 ml/ min. Sample compartment temperature was maintained at 48C and the column was maintained at ambient temperature. DB-67 carboxylate and lactone standards were prepared in 10 mm sodium carbonate buffer (ph ¼ 10.3) and methanol, respectively. The retention times were JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY and 5.2 min for DB-67 carboxylate and lactone, respectively. Lipid analyses were performed using HPLC coupled to an evaporative light scattering detector (ELSD, Sedere, Inc., Lawrenceville, NJ). Separations employed an Allsphere (Alltech Associates, Inc., Deerfield, IL) silica column (5 mm, mm) without a guard column and a linear gradient, starting with 100% (v/v) mobile phase A (80% chloroform:19.5% methanol:0.5%(v/v) NH 4 OH) changing to 80% mobile phase A:20% mobile phase B (80% methanol:19.5% water:0.5% (v/v) NH 4 OH) at 3 min. The gradient was maintained at 80% A:20% B till 7 min and changed back to 100% A by 14 min. The total run time was 15 min at 1 ml/min. ELSD settings included a gain of 10, temperature of 408C and a pressure of 2.2 lb. Sample compartment temperature was maintained at 48C and the column was maintained at ambient temperature. The retention time for DSPC was approximately 7.5 min (Fig. 1). Response factor of the standards (prepared in mobile phase A in the range of mg/ml) was calculated using log concentration and log peak area. Samples collected for lipid analysis during partitioning or release experiments were immediately transferred to test tubes, dried under nitrogen, and the resulting lipid film was stored at 258C until analysis. The lipid films were reconstituted in ml of mobile phase A for analysis. DATA ANALYSIS Mathematical Model for Drug Efflux from Liposomes in Aqueous Buffer The permeability coefficient for DB-67 transport across the DSPC lipid bilayer at 378C was obtained from dynamic dialysis of liposome suspensions containing entrapped DB-67 versus blank liposomes spiked with DB-67 (Scheme 2). Samples taken from inside the dialysis tube at various times were analyzed for total drug remaining. The total mass of DB-67 (M d ) in the dialysis tube at any given time is: M d ¼ M i þ M o (2a) where M i and M o are masses of DB-67 inside and outside the liposomes. The total volume of liposome suspension in the dialysis tube, V d, is: V d ¼ V i þ V o (2b) DOI /jps

6 LIPOSOME TRANSPORT OF HYDROPHOBIC DRUGS 405 Figure 1. Representative chromatogram obtained during analysis of DSPC by HPLC with ELSD detection. where V i is the total liposomally entrapped aqueous volume plus the lipid volume in the inner bilayer leaflet of vesicles in the suspension and V o is the extravesicular aqueous volume along with the volume of lipid located in the outer leaflet of suspended liposomes in the dialysis tube. For a total number of vesicles, n, V i and V o can be expressed as: V i ¼ nðv w i þ V m i Þ (2c) V o ¼ nv m o þ V w o (2d) where Vi w and Vi m are the volumes of the inner aqueous compartment and inner monolayer in each vesicle, respectively, Vo m is the volume of the outer monolayer in each vesicle and Vo w is the extravesicular aqueous volume in the dialysis tube. Converting masses in Eq. (2a) to corresponding concentrations, M d V d ¼ M i V d þ M o V d L ¼ M i þ M o V d ffi V o ; x ¼ V o xv i V o V i (2e) (2f) Scheme 2. Schematic depicting liposome loaded (Panel A) versus liposome spiked (Panel B) experiments used in DB-67 (represented by D) permeability studies. Rate constants k m and k d are the rate constants for permeation across the bilayer membrane and dialysis tube membrane respectively. L ¼ L i x þ L o (2g) where, L is the total suspension concentration of DB-67 in the dialysis tube, L i and L o are internal and external system concentrations of DB-67, and x is the ratio of the volume outside and inside the vesicles, where, again, the volume of the inner bilayer leaflet is included as a component of the internal volume while the outer bilayer leaflet contributes to the external volume. The total internal mass of DB-67, M i, can be related to the masses of drug in the liposome aqueous core (Mi w ) and bound to the inner monolayer (Mi m ) which provides the following DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008

7 406 JOGUPARTHI, XIANG AND ANDERSON expression after dividing by V i : M i V i ¼ Mw i V i þ Mm i V i Eq. (3a) can also be expressed as: L i ¼ Lw i a þ Lm i a ¼ V i b nvi w ; b ¼ V i nvi m ; L w i ¼ Mw i nvi w ; L m i ¼ Mm i nvi m (3a) (3b) where a and b are the ratios of total internal volume of the vesicles to the total entrapped aqueous core volume and total volume of lipid in the inner leaflets. Defining K p, the volume-based partition coefficient, as: K p ¼ Lm i L w i (3c) The concentration of drug in each intravesicular compartment can be described: L w i ¼ abl i b þ ak P ¼ a i L i (3d) L m i ¼ abk PL i ¼ b b þ ak i L i (3e) P where L w i and L m i are concentrations of drug in the liposome aqueous core and inner monolayer, respectively, and a i and b i are factors to convert the total internal system concentration (L i )tothe respective drug concentrations in the aqueous and membrane phases. The fractions of free and membrane bound drug inside the vesicles are a i / a and b i /b, respectively. The parameters a and b are dependent on vesicle size and bilayer thickness but independent of the lipid concentration. The value of a approaches one (b!1) with increasing vesicle size since the thickness of the membrane becomes negligible compared to the total diameter. Similar to the above treatment, the concentrations of drug in the external aqueous compartment within the dialysis tube (L w o ) and in the outer monolayer (L m o ) are: L w o ¼ cdl o d þ ck P ¼ a o L o L w o ¼ Mw o V w o L m o c ¼ V o ; L m i ¼ Mm o nv m o V w o ¼ cdk PL o d þ ck P ¼ b o L o ; d ¼ V o nvo m ; (4a) (4b) where c and d are the ratios of total external volume to that of the external aqueous volume and the outer monolayer volume, respectively, and a o and b o are multiplying factors to obtain the respective drug concentrations in the external aqueous and membrane phases from the total external concentration within the dialysis tube. The fractions of free and membrane bound drug in the external compartment are a o /c and b o /d, respectively. The parameters c and d are dependent on vesicle size, bilayer thickness, and the lipid concentration in the suspension. The value of c approaches one (d!1) in dilute liposome suspensions since the entrapped volume becomes negligible compared to the total suspension volume. The solute flux across a homogeneous membrane derived from Fick s first law is: dm dt ¼ D mk m=w A ðl w i L w o h Þ (5a) m where D m is the diffusion coefficient within the membrane, K m/w is the membrane/water partition coefficient, and A and h m are the membrane area and thickness, respectively. (Although lipid bilayer heterogeneity implies that a more complex treatment than that in Eq. (5a) is necessary, the barrier-domain solubility-diffusion model for lipid bilayer permeability attempts to circumvent this complication by assuming that the barrier properties of lipid bilayers reflect a nearly homogeneous sub-domain (e.g., the ordered hydrocarbon chain region; vide infra). Under these conditions, the diffusion coefficient, partition coefficient and membrane thickness in Eq. (5a) refer to properties of the barrier domain.) Eq. (5a) can also be expressed in terms of the permeability coefficient, P m ðd m K m=w =h m Þ: dm dt ¼ P maðl w i L w o Þ (5b) Mass flux can be converted to the corresponding change in internal liposomal drug concentration: dl i dt ¼ P ma ðl w i L w o V Þ (5c) i dl i dt ¼ k mða i L i a o L o Þ (5d) where k m, the first-order rate constant for efflux, can be used to calculate P m. k m ¼ P ma V i ffi 3P m R (5e) JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008 DOI /jps

8 LIPOSOME TRANSPORT OF HYDROPHOBIC DRUGS 407 Similarly, the rates of change of external drug concentration (L o ) within the dialysis tube and drug concentration (L b ) in the reservoir in which the dialysis tube is immersed having a volume V bulk are: dl o dt ¼ k m x ðlw i L w o Þ k dl w o x ¼ V o (6a) V i dl o dt ¼ k m x ða il i a o L o Þ k d ða o L o Þ dl b dt ¼ k d y ða ol o Þ y ¼ V bulk V d (6b) (6c) where k d is the first-order rate constant for drug permeation across the dialysis membrane. Eqs. (6a 6c) assume sink conditions in the reservoir (i.e., drug concentration in the reservoir is negligible in comparison to the external drug concentration within the dialysis tube (L b << L w o )) such that back flux of drug into the dialysis tube can be ignored. Eqs. (5d) and (6b) can be solved using Laplace transforms to obtain the following general solutions: L i ¼ L ið0þ H G a ok d þ k m x a o G e Gt a o k d þ k (7a) m x a o H e Ht L o ¼ L ið0þa i k m xðh GÞ ½e Gt e Ht Š ðh 6¼ GÞ (7b) L o ¼ L o (0)) liposome suspensions by non-linear least squares regression analysis (Scientist 1, Micromath Scientific Software, St. Louis, MO). The volume ratios (a, b, c, d) used to calculate the fractions of drug bound inside and outside the vesicles were obtained from the lipid concentration and the particle size of the liposomes (after calculation of liposome entrapped and free volume, see Eqs. 3b and 4a). Since literature studies employing dynamic dialysis to monitor drug release from liposomes or nanoparticles often assume a simple first-order rate constant for drug efflux from the dialysis tube or drug appearance in the reservoir, it is of interest to explore the experimental conditions or parameter values under which the above solutions reduce to simple first-order behavior. At steady-state ðdl o =dt ffi 0Þ, the ratio of the external drug concentration within the dialysis tube to that inside the vesicles can be expressed as: L o L i ¼ k m a i ðk m þ xk d Þa o (8a) Substituting Eq. (8a) into Eq. (5d) and simplifying gives: dl i dt ¼ k dk m xa i L i ðk m þ xk d Þ (8b) If the rate constant for drug transport across the dialysis membrane is much faster than back flux into the liposomes (k d >> k m =x), Eq. (8b) can be further simplified: q G ¼ a ik m þ a o k d þ k ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi m x a o þ a i k m þ a o k d þ k m 2 4ai x a o a o k m k d 2 ð7cþ H ¼ a ia o k m k d G (7d) dl i dt ¼ k ma i L i ¼ k app L i (8c) LðtÞ ¼ L i x þ L o (7e) In the present experiments, the total drug concentration versus time profiles inside the dialysis tube [L(t)] initially containing either drug-loaded liposomes or blank liposomes with drug spiked into the external aqueous compartment were fit to Eq. (7e) subject to the revelant initial conditions for drug-loaded (At t ¼ 0, L i ¼ L i (0); L o ¼ 0) or spiked (At t ¼ 0, L i ¼ 0; dl i dt ¼ 1 dl i x dt ¼ k appl (8d) Under these conditions, the apparent permeability coefficient ( P app ) generated from the apparent first-order rate constant (k app ) can be used to calculate the true permeability coefficient ( P m ): P app ¼ fub i P m fub i ¼ a i a ; a ffi 1 (8e) DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008

9 408 JOGUPARTHI, XIANG AND ANDERSON Thus, under the appropriate conditions (i.e.,dl o =dt ffi 0; k d >> k m =x), an accurate liposomal membrane permeability coefficient can be obtained from the apparent rate constant for drug efflux from the dialysis tube after correction for drug binding to the inner monolayer. The conditions under which Eq. (8d) could be employed were explored quantitatively by generating drug concentration-time profiles in the dialysis tube using Eq. (7e) then applying Eqs. (8d) and (8e) to obtain a value for P m that could be compared to the original value used in the simulations. Obtaining a reliable value for P m by dynamic dialysis requires that the rate of drug disappearance from the dialysis tube be governed by the apparent rate constant for release from the vesicles (k app < k d ). This condition inherently puts an upper limit on the maximum apparent permeability coefficient that can be obtained by this method as P app < R v k d =3cm=s, where R v is the radius of the vesicle. At steady-state and when back flux of drug into the liposomes can be ignored (i.e.,dl o =dt ffi 0; k d >> k m =x), the maximum membrane permeability coefficient that can be determined using the dynamic dialysis method is: P m < R vk d 3a i (8f) Upon substitution of the relevant volume terms for a i, and simplifying, this upper P m value can be expressed as a function of vesicle radius, permeant partition coefficient, bilayer thickness and the rate constant for dialysis tube permeation: P m < k dr v ½R 3 v ðr v h m Þ 3 þ 2K p ðr v h m Þ 3 Š 3½R 3 v þðr v h m Þ 3 Š (8g) Mathematical Model for DB-67 Efflux from Liposomes and Hydrolysis in Plasma Since the volume of liposomes added to plasma was small compared to the total volume of plasma and both DB-67 lactone and carboxylate bind to plasma proteins with high affinity, 7,8,10,11 sink conditions were assumed for drug released from liposomes into plasma. Due to the low ph inside the liposomes (ph ¼ 4) and the ability of citrate buffer to maintain the trans-membrane ph gradient in plasma (ph ¼ 7.4) for the time period of this study, all of the entrapped DB-67 was assumed to exist in lactone form. Carboxylate was JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008 assumed to form only from DB-67 lactone after its release into plasma. Rate equations for the liposomal and plasma lactone and carboxylate concentrations are then: dl i dt ¼ k0 m ðlw i L w o Þ (9a) dl o dt ¼ k0 m x ðlw i L w o Þ k ol o þ k c C o (9b) dc o dt ¼ k ol o k c C o (9c) where k 0 m is the first-order rate constant for transport of the lactone from liposomes into plasma, L i and L o are the concentrations of lactone inside liposomes and in plasma, respectively, C o is the concentration of carboxylate in plasma, and k o and k c are the rate constants for lactone ring-opening and closing in plasma, respectively. The kinetics of hydrolysis of drug added directly to plasma could also be described by Eq. (9b) (excluding the liposome transport term) and (9c). Data from the hydrolysis kinetics of liposomally entrapped DB-67 and nonliposomal DB-67 were fit simultaneously to Eqs. (9a 9c) to obtain estimates of k 0 m, k o, and k c by nonlinear leastsquares regression analysis. The liposomal permeability of DB-67 in plasma could then be obtained from k 0 m using Eq. (5e). RESULTS Validation of Analytical Methods and Characterization of Liposomes DB-67 samples from size exclusion chromatography, partitioning studies and dynamic dialysis studies were analyzed by HPLC with fluorescence detection. Initial DB-67 concentrations were in the range of and nm in the dynamic dialysis experiments and bilayer/water partition coefficient determinations, respectively. The HPLC response was found to be linear from 5 to 30 nm for DB-67 lactone so all samples were diluted in acidified methanol to a lactone concentration within this range. Coefficients of variation in the response factors were 5% intraday and 6% interday. The initial concentrations of DB-67 in plasma hydrolysis experiments ranged from 50 to 300 nm. The HPLC response factor for DB-67 carboxylate was linear over the range nm. Plasma DOI /jps

10 LIPOSOME TRANSPORT OF HYDROPHOBIC DRUGS 409 extracts were directly analyzed by HPLC or further diluted with cold methanol acetonitrile where necessary to be within this range. Precision of DB-67 carboxylate response factors was 3% intraday (5% interday). The extraction efficiency of DB-67 in spiked plasma samples using DB-67 concentrations of nm was >95% and the lactone carboxylate interconversion quenching efficiency was 100% (Fig. 2). Liposomes prepared for these studies exhibited particle diameters of nm obtained by DLS. Particle diameter and ph of vesicle suspensions were constant after preparation and during storage. For both dynamic dialysis and plasma hydrolysis experiments, liposome loaded drug was separated from free drug by size exclusion chromatography while monitoring light scattering intensity (kilocounts/s (Kcts)) of eluent fractions. Representative elution profiles for DB-67 and drug-loaded liposomes are shown in Figure 3. Liposomes eluted between ml and were well separated from the free drug. Figure 3. Elution profiles of free (~) or liposome loaded DB-67 (~) as determined by HPLC (nm) and liposomes (&) as determined by light scattering intensity (kilocounts/s (Kcts)). The lipid concentration in liposome suspensions was 0.16 mg/ml during dynamic dialysis studies (after Sephadex 1 elution and dilution) and mg/ml during plasma hydrolysis experiments. Lipid concentration was analyzed during bilayer/water partition studies and bilayer transport studies by gradient HPLC with ELSD detection. The response factor (RF) was nonlinear within the fourfold concentration range of the standards employed. Therefore a log concentration versus log peak area was used to calculate the response factor. Precision of the assay was 6% intraday and <8% interday. Since DSPC composition was 95% of the total lipid in the vesicles and given the similar chain length of both DSPC and DSPE, the vesicles were assumed to be 100% DSPC for estimation of volume parameters. Previously reported 36 values for bilayer and head group thickness of DSPC vesicles were used to calculate volume ratio parameters (a, b, c, d, x) for the estimation of permeability coefficients. Figure 2. Extraction of DB-67 from plasma at varying DB-67 concentration (Panel A) and the quenching efficiency of the reaction between DB-67 lactone and carboxylate (Panel B) starting from either lactone or carboxylate as the initial reactant. 1, 9-Decadiene and Lipid Bilayer/Water Partition Coefficients A 1, 9 decadiene/water partition coefficient for DB-67 of (n ¼ 4) was found. The concentration dependence of this value was not explored due to the low solubility of DB-67 both in decadiene and ph 4 acetate buffer. 16 Figure 4 shows the partition coefficient of DB-67 determined by equilibrium dialysis as a function of lipid concentration. The drug concentration could not be varied over a wide range in the partition experiments here due the low intrinsic solubility of DB Within the concentration range used ( nm), the partition coefficient DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008

11 410 JOGUPARTHI, XIANG AND ANDERSON Figure 4. Membrane (DSPC with 5mol% m-peg DSPE) partition coefficient of DB-67 (Mean SD) as a function of varying lipid concentration. Figure 5. Concentration of DSPC (&) in the dialysis tube during transport experiments. DSPC was analyzed by a gradient HPLC method with ELSD detection. was found to be and independent of drug and lipid concentration. Equilibrium was attained within 24 h and lipid analysis indicated that the concentration of DSPC remained constant over this period. Efflux of DB-67 from Liposomes in Aqueous Buffer Initial studies of DB-67 permeability were conducted using a previously developed ultrafiltration technique However this method was found to be unsuitable due to significant adsorption of DB-67 lactone to ultrafilter membranes (data not shown) and the inability to maintain sink conditions in a reasonable extravesicular volume. The dynamic dialysis method developed involves monitoring the liposome suspension concentration of DB-67 within the dialysis tube as a function of time while dialyzing the suspension against a large reservoir of buffer to maintain sink conditions and ensure complete release of entrapped drug. Initially, the reservoir solution was magnetically stirred during the transport experiments but significant increases in lipid concentration within the dialysis tube were observed, reflecting an apparent reduction in fluid volume presumably due to an increase in fluid pressure on the dialysis tube during stirring. A decrease in stirring rate did not completely ameliorate fluctuations in lipid concentration. In the absence of stirring, the lipid concentration in the dialysis tube remained constant during the transport experiments (Fig. 5). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008 Figure 6 shows the fractions of initial DB-67 lactone (mean SD) remaining in the liposomal suspension within the dialysis tube at various times after introducing liposomes containing varying concentration of entrapped DB-67 or spiking blank liposome suspensions with DB-67. The apparent half-life for loss of liposomally entrapped DB-67 lactone (based on the decline in drug concentration inside the dialysis tube) was h compared to h for DB-67 added to blank liposomes. This 6-fold difference did not appear to be sufficiently large to justify the Figure 6. Fractions of initial amount of DB-67 lactone (mean SD, n ¼ 4) remaining inside the dialysis tube in liposomes spiked with drug (*) or liposomes entrapped with drug (&). The solid line is a fit of the data generated using Eqs. (7a 7e). The initial DB-67 concentration (entrapped) of the liposome suspension ranged from 50 to 2700 nm. DOI /jps

12 LIPOSOME TRANSPORT OF HYDROPHOBIC DRUGS 411 steady-state assumption. Therefore, the complete transport model developed previously (Eq. 7e and supporting equations) was used to simultaneously fit the two concentration-time profiles. Table 1 lists the estimated phase volume ratios and other constants that were used in the regression analyses. The solid lines shown in Figure 6 represent the fitted profiles for the fraction of DB-67 lactone remaining in the dialysis tube from which values for k m and k d, the first-order rate constants for DB-67 transport across the liposome and dialysis membranes, respectively, were generated. At the lipid concentration employed (0.16 mg/ml), greater than 99% of the entrapped drug is bound to the inner bilayer leaflet. Using the fitted value for k m, the membrane permeability coefficient for DB-67 lactone was found to be cm/s. Based on the experimentally measured value for K p, about 13% of the drug released from liposomes but remaining within the dialysis tube is bound to the outer monolayer. This was considered in the mathematical model used to fit the data in Figure 6. Using the value for k m, the half-life for liposomal release of DB-67 under sink conditions (conditions in which drug binding to the outer monolayer of liposomes is negligible) can be estimated to be h. Similarly, the half-life for release of DB-67 from the dialysis tube under sink conditions and in the absence of perturbing effects from liposomes in the dialysis tube can be estimated from k d to be h. Table 1. Estimated Phase Volume Ratios and other parameters for the Lipid Concentration used in the Transport Experiments in Buffers and Plasma Parameter Buffer Plasma n a b c 1 1 d x K p Suspension lipid concentration 0.16 mg/ml mg/ml Parameters a and b are the ratios of the total intravesicular volume to that of the aqueous core and inner lipid monolayer volumes, respectively. Parameters b and c are the ratios of the total extravesicular volume in the dialysis tube to that of the aqueous and outer lipid monolayer volumes, respectively. n is the number of vesicles, K p is the volume partition coefficient and x is the ratio of total unentrapped to entrapped volume. Table 2 displays the calculated values of k m, the corresponding liposomal membrane permeability coefficient of DB-67, and k d as a function of drug concentration over a concentration range of nm. Within this concentration range, the transport parameters were observed to be independent of drug concentration. Due to the low solubility of DB-67 lactone, 16 formulation of liposomes at higher encapsulated drug concentrations was not feasible. Kinetics of DB-67 Lactone Release from Liposomes and Hydrolysis in Rat Plasma The kinetics of DB-67 release from liposomes were also evaluated by comparing the hydrolysis kinetics of free versus liposome encapsulated DB-67 in rat plasma. Figure 7 shows the observed lactone and carboxylate concentration (mean SD) versus time profiles generated after a liposome suspension containing entrapped DB-67 or free DB-67 lactone were added to plasma. The data in Figure 7 were fit simultaneously (Eqs. 9a 9c) assuming that hydrolysis of DB-67 lactone to DB-67 carboxylate occurs after lactone release from liposomes. The assumption that the liposomally entrapped species is the lactone form over the 12 h period during which hydrolysis was monitored is supported by the fact that citrate buffer has been shown to maintain a low ph inside liposomes for more than 24 h in vivo. 38 The curves in Figure 7 represent the best fits of Eqs. (9a 9c) to the experimental data. From the value for k 0 m, the liposome permeability coefficient for DB-67 lactone was found to be cm/s in plasma, which is not significantly different from that observed in aqueous buffers. The rate constants for DB-67 lactone ringopening and ring-closure in plasma (ph 7.4) were observed to be and h 1, corresponding to a half-life to equilibrium for ring opening of DB-67 of min which is slightly higher than that in PBS ( min) but much lower than that in whole blood ( min). 1 The half-life to equilibrium for DB-67 lactone ring-opening is much greater than that for camptothecin in rat plasma (4.6 min). 39 This is consistent with the hypothesis that blood stability of DB-67 lactone is improved compared to camptothecin due to differences in protein binding and partitioning into red blood cell membranes. DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008

13 412 JOGUPARTHI, XIANG AND ANDERSON Table 2. Estimated Rate Constants for Lipid Bilayer and Dialysis Membrane Transport ( 95% CI) of DB-67 in Dynamic Dialysis Experiments and the Intrinsic Permeability Coefficients for Bilayer Membrane Transport of DB-67 Obtained from Application of the Model Described in Eqs. (7a 7e) at Varying Concentrations of DB-67 Initial DB-67 Concentration in Liposome Suspension (nm) k m (h 1 ) k d (h 1 ) P m (10 8 ) cm/s DISCUSSION Figure 7. Concentration-time profiles (n ¼ 4) for the disappearance of DB-67 lactone (Mean SD) when entrapped in liposomes (&) or as free drug (D) and the appearance of DB-67 carboxylate (mean SD) in liposome entrapped (&) or free (~) plasma hydrolysis experiments. The solid lines were generated by fitting the concentration-time data to Eqs. (9a 9c). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008 Liposomal Formulation of Neutral and Weakly Acidic Amphiphiles The therapeutic class of camptothecins consists both of hydrophobic compounds that are unionized in their lactone form but weakly acidic after lactone ring-opening (DB-67, karenitecin, SN-38, 9-nitro camptothecin etc.) and weakly basic amines (topotecan, irinotecan, lurtotecan, exatecan, etc.). The active form of the drug is the neutral lactone form and it is desirable to deliver the active form of the drug to tumor tissue. Liposomes are being considered for the formulation of a variety of anticancer agents following the clinical success of liposomal doxorubicin Success of liposomal formulations relies on the ability to load drugs at therapeutically relevant concentrations and retain the encapsulated drug in liposomes while in the circulation. Most formulation techniques developed thus far for encapsulation of anti-cancer agents including camptothecins are active loading methods (e.g., ph, ion or ammonium sulfate gradient methods) modeled after methods that were useful for loading doxorubicin, an amphiphilic amine. The successful retention of amphiphilic amines entrapped by these active loading methods has been due to trans-membrane ph gradients that are retained in vivo (low intraliposomal ph) or intraliposomal drug or salt precipitation that results in improved retention. 28,38,43 47 Liposome loading and retention strategies developed for amphiphilic amines allow for the loading and retention of the active lactone form of cationic (i.e., weakly basic amine-containing) camptothecins but such techniques are not useful for neutral analogues. For example, DB-67 has been previously loaded into DMPC vesicles using a passive encapsulation technique that relies on the binding of the lactone form of the drug to the liposome membrane but the pharmacokinetic profile for DB-67 when administered in DMPC liposomes did not differ significantly from that of the free drug due to the rapid leakage of encapsulated drug (Zamboni et al., Unpublished work). Two techniques that have been proposed recently for liposomal loading of neutral compounds and weak acids include attempts to entrap drug-cyclodextrin (CD) complexes and drug loading in the presence of a trans-membrane ph gradient combined with a metal ion (Na þ,ca 2þ ) gradient. 51 Both techniques have shown improved drug loading but have failed to prolong liposome retention to the extent that has been possible DOI /jps

14 LIPOSOME TRANSPORT OF HYDROPHOBIC DRUGS 413 with amphiphilic amine-containing compounds. The lack of success of the drug-cd entrapment strategy has been attributed to lipid bilayer destabilization by entrapped cyclodextrin ,52,53 Thus, in spite of the demonstration of excellent anti-cancer activity by several neutral or weakly acidic hydrophobic compounds, active liposomal loading and retention strategies have not been successful for this group of compounds. In the present studies in plasma, a prolonged liposomal retention half-life of approximately 3 h (Fig. 7) has been observed by encapsulation of DB- 67 in rigid gel phase DSPC bilayers. Given this substantial improvement in retention, it would be of interest to ascertain whether or not this value could have been anticipated and whether or not retention could be prolonged further. The Barrier-Domain Solubility Diffusion Model Estimate of the Permeability Coefficient of DB-67 The success of formulation-based approaches for improving the liposome loading and retention of drug candidates relies to a large extent on altering the rate of bilayer permeation of the encapsulated material (drug plus excipients). Systematic selection and manipulation of variables such ph, buffers, lipid composition and other excipients may enable one to control the retention of neutral and weakly acidic hydrophobic compounds. If the effects of these variables on bilayer permeability could be fully understood it may be possible to predict the permeability of a given compound under various conditions. Previous studies in these and other laboratories have investigated the role of membrane composition and ph on the bilayer permeability of weak acids and predictive relationships have been developed to account for the effect of varying ph and bilayer phase structure on permeability ,36,54 Approaches to predict passive permeability across lipid bilayers and biomembranes based solely on the structure of the permeant and compositions of the membrane have been the subject of research in biology for over a century. The simplest model is the bulk-solubility diffusion model which assumes that the membrane is homogenous and isotropic. The permeability coefficient derived from this model is: P o ¼ PC m=wd m (10a) h m where P o is the permeability coefficient, PC m/w is the membrane/water partition coefficient, h m is the thickness of the membrane and D m is the diffusion coefficient. Taking into account the heterogeneity of the bilayer, Diamond and Katz 55 proposed a general expression for the passive permeability of a solute across a membrane: Z 1 ¼ r 0 dx þ P m KðxÞDðxÞ þ r00 (10b) where P m is the permeability coefficient, K(x) and D(x) are the local partition and diffusion coefficients at a depth x normal to the bilayer and r 0 and r 00 are interfacial resistances. Xiang and Anderson further simplified the above model by assuming that permeability across a bilayer may be rate-limited by a distinct region (barrier-domain) within the bilayer. 33 The barrier domain was shown to exhibit a chemical selectivity similar to that expected for the hydrocarbon chain region in liquid crystalline bilayers though its properties vary somewhat with the lipid bilayer phase structure. 31,36,54,56 Xiang and Anderson 31 33,36,56,57 also developed a model to account for the effects of bilayer chain ordering as well as permeant size on the permeability. The permeability coefficient according to the barrierdomain solubility diffusion mode is: 54 P m ¼ K barrier=waterd barrier h barrier ¼ fp o (10c) where K barrier/water is the barrier/water partition coefficient of the solute, h barrier is the thickness of the bilayer chain region, and D m is the diffusion coefficient. P m has been shown to be equal to the product of permeability coefficient, P o from the homogeneous bulk solubility diffusion theory and a scaling factor f, the permeability decrement due to lipid chain ordering. The scaling factor f was shown to be dependent on the two-dimensional packing structure, as characterized by the free surface area per lipid molecule, a f, and the solute size parameter, a s : 56 f ¼ f o exp la S (10d) a f where a s is the minimum cross sectional area of the solute and f o and l are constants independent of permeant size and bilayer packing structure. Depending on the composition of the membrane, the chemical selectivity of the barrier-domain has been shown to be mimicked by suitable reference solvents, exemplified by 1,9-decadiene in the case of phosphatidylcholine and egg DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008